Methods, devices and kits for peri-critical reflectance spectroscopy

ABSTRACT

Spectroscopy apparatuses oriented to the critical angle of the sample are described that detecting the spectral characteristics of a sample wherein the apparatus consists of an electromagnetic radiation source adapted to excite a sample with electromagnetic radiation introduced to the sample at an angle of incidence at or near a critical angle of the sample; a transmitting crystal in communication with the electromagnetic radiation source and the sample, the transmitting crystal having a high refractive index adapted to reflect the electromagnetic radiation internally; a reflector adapted to introduce the electromagnetic radiation to the sample at or near an angle of incidence near the critical angle between the transmitting crystal and sample; and a detector for detecting the electromagnetic radiation from the sample. Also, provided herein are methods, systems, and kits incorporating the peri-critical reflectance spectroscopy apparatus.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/025,737, filed Feb. 1, 2008, which application is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Internal reflection spectroscopy, also known as Attenuated TotalReflectance (ATR) spectroscopy, has been know for many years, and is awidely used method of sampling in infrared (IR) and fluorescencespectroscopy, as well as in other spectroscopies. Mid-wavelengthinfrared (MWIR), or intermediate infrared (IIR), spectroscopy has overthe years become a technique of choice when specificity is of utmostimportance. It has historically been a difficult technique to use forseveral reasons. First, absorptivities of many materials are quite highin the mid-wavelength infrared region of the electromagnetic spectrum(e.g., from about 3-8 μm) While this is good from the standpoint ofsensitivity, it makes sampling sometimes complex. As a result, a widevariety of sampling technologies have been developed to help introducethe sample to the spectrometer in an ideal fashion. A ubiquitous andproblematic sample component is water. In the near-infrared (NIR)region, using wavelengths from about 800 nm to 2500 nm, another problemthat can arise is the fact that the path length may be too short. Oneadvantage is that near-infrared can typically penetrate much fartherinto a sample than mid infrared radiation.

One problem faced when using spectroscopy is the fact that many samplepreparations contain water. Water has a very high absorbance in themid-infrared. Therefore, in order to measure a spectrum of water in theclassical mid-infrared region of 4000-400 cm ⁻¹, the path length must belimited to less than a few 10 s of microns. ATR can provide this verysmall path length needed. In other situations however, the path lengthof ATR is too small for ideal sampling. This can be the main problemwhen trying to make measurements through mammalian skin or otherbiological tissue, or when the desired spectral information is from adeeper depth and not adjacent the surface of the mammalian skin.

Attenuated Total Reflectance (ATR) is often indicated in difficultsampling situations. The spectroscopic usefulness of the effect wasfirst noticed in the 1960s by Fahrenfort and is predictable from basicoptical physics. Basically, when light propagates through a medium ofhigh refractive index and approaches an interface with a material oflower refractive index, a transmission and a reflection will occur. Therelative strengths of these transmissions and reflections are governedby the Fresnel equations:

$\begin{matrix}{{r_{\bot} \equiv \frac{E_{r}}{E_{i}}} = \frac{{\frac{n_{1}}{\mu_{1}}\cos \; \theta} - {\frac{n_{2}}{\mu_{2}}\cos \; \theta^{\prime}}}{{\frac{n_{1}}{\mu_{1}}\cos \; \theta} + {\frac{n_{2}}{\mu_{2}}\cos \; \theta^{\prime}}}} & (1) \\{{t \equiv \frac{E_{t}}{E_{i}}} = \frac{2\frac{n_{1}}{\mu_{1}}\cos \; \theta}{{\frac{n_{1}}{\mu_{1}}\cos \; \theta} + {\frac{n_{2}}{\mu_{2}}\cos \; \theta^{\prime}}}} & (2) \\{{r_{||} \equiv \frac{E_{r}}{E_{i}}} = \frac{{\frac{n_{2}}{\mu_{2}}\cos \; \theta} - {\frac{n_{1}}{\mu_{1}}\cos \; \theta^{\prime}}}{{\frac{n_{1}}{\mu_{1}}\cos \; \theta^{\prime}} + {\frac{n_{2}}{\mu_{2}}\cos \; \theta}}} & (3) \\{{t_{||} \equiv \frac{E_{t}}{E_{i}}} = \frac{\mu_{1}}{{\frac{n_{1}}{\mu_{1}}\cos \; \theta^{\prime}} + {\frac{n_{2}}{\mu_{2}}\cos \; \theta}}} & (4)\end{matrix}$

The Fresnel equations give the ratio of the reflected and transmittedelectric field amplitude to initial electric field for electromagneticradiation incident on a dielectric.

In general, when a wave reaches a boundary between two differentdielectric constants, part of the wave is reflected and part istransmitted, with the sum of the energies in these two waves equal tothat of the original wave. Examination of these equations reveals thatwhen the light is traversing through a high index medium and approachingan interface with a low index medium, the reflected component can betotal, with no light being transmitted. The angle at which this occursis called the critical angle and is defined by the following equation(5):

$\begin{matrix}{\theta_{C} = {\sin^{- 1}\left( \frac{n_{2}}{n_{1}} \right)}} & (5)\end{matrix}$

The reflected component has an angle of reflection equal and opposite tothe angle of incidence upon the interface. Above the critical angle, alllight is reflected. Below the critical angle, some light would transmitthrough the interface according to the above Fresnel equations. A deviceoperating in this mode would use light that refracts according toSnell's Law (equation (6)):

n ₁ sin θ=n₂ sin θ′  (6)

As previously stated, above the critical angle reflection is total.Fahrenfort first noticed that upon total reflection, a standing, orevanescent, wave is set up at the interface between high and low index.The wave has an exponentially decaying intensity into the rarer (lowerindex) medium. If an absorbing substance is placed in the vicinity ofthis evanescent (standing) wave, which extends a distance into the rarermedium, it can absorb portions of the light in specific wavelengthscorresponding to the absorption properties of the material. In this way,the total reflection is said to be “frustrated” by the absorption of thesample. The returning light at the detector then is evaluated todetermine the missing energy. It follows that this mode can be used toobtain an infrared spectrum of a material in contact with the high indexmedium through which the light is traveling. The strength of thisinteraction can be predicted through several equations developed byHarrick. First, the depth of penetration is defined as the 1/e point ofthe exponential decay of the evanescent (standing) wave (equation (7)):

$\begin{matrix}{d_{p} = \frac{\lambda/n_{1}}{2{\pi \left( {{\sin^{2}\theta} - \left( \frac{n_{2}}{n_{1}} \right)^{2}} \right)}^{\frac{1}{2}}}} & (7)\end{matrix}$

where n₂ is the sample refractive index and n₁ is the crystal refractiveindex. The depth of penetration is defined as the point at which thestrength of the evanescent wave electric vector decays to a value of 1/e(where e is Euler's number) from its original strength. Quickcalculations are often done using the depth of penetration tocharacterize the strength of signal that will be obtained with ATR. Thequick calculations may be less accurate but are suitable for providing aguide. A more accurate equation for the point where the evanescent waveelectric vector decays was derived by Harrick, namely the effectivethickness or effective depth, d_(e).

An additional complication arises if the sample is thin compared to the1/e point of the evanescent wave. The effective thickness calculationresults in a number that can be used in Beer's Law calculations, and isclosely related to the path length in a transmission measurement made atnormal incidence. There are now three refractive indices to worry about:n₁, the index of the crystal, n₂, the index of the thin layer of sample,and n₃, the index of whatever is beyond the sample, usually air. Also,since the geometry is usually not near-normal, the calculation must bedone for three orthogonal axes. Finally, the measurement is polarizationdependent and should be calculated for two orthogonal polarizations. Forpurposes of this discussion, the thin layer is assumed to by isotropicand the polarization is deemed to be random. So the effective depthequation, for thin layers of sample where the sample layer thickness ismuch less than the depth of penetration, is as follows:

$\begin{matrix}{d_{e} = {\frac{1}{\cos \; \theta}\frac{n_{2}}{n_{1}}\frac{d_{p}}{2}{E_{02}^{r\; 2} \cdot \left( {{\exp \left( {- \frac{2z_{i}}{d_{p}}} \right)} - {\exp \left( {- \frac{2z_{f}}{d_{p}}} \right)}} \right)}}} & (8)\end{matrix}$

where the z values are the initial and final z-dimension positions ofthe film relative to the surface of the ATR prism. The E term is thesquare of the strength of the electric vector in medium 2 E isproportional to light intensity. For polarized incident light

E_(02,∥) ^(r2)=E_(02,x) ^(r2)+E_(02,z) ^(r2)   (9)

and

E_(02,⊥) ^(r2)=E_(02,y) ^(r2)   (10)

and this results in

d _(e,∥) =d _(ex) +d _(ez)   (11)

and

d_(e,⊥)=d_(ey)   (12)

and

d _(e,random)=(d _(e,⊥) +d _(e,∥))/2   (13)

The three orthogonal electric field components are calculated fromFresnel's equations:

$\begin{matrix}{E_{{0x},2}^{r} = \frac{2\cos \; {\theta \left( {{\sin^{2}\theta} - n_{31}^{2}} \right)}^{1/2}}{{\left( {1 - n_{31}^{2}} \right)^{1/2}\left\lbrack {{\left( {1 + n_{31}^{2}} \right)\sin^{2}\theta} - n_{31}^{2}} \right\rbrack}^{1/2}}} & (14) \\{{E_{{0z},2}^{r} = \frac{2\cos \; {\theta sin}\; \theta \; n_{31}^{2}}{{\left( {1 - n_{31}^{2}} \right)^{1/2}\left\lbrack {{\left( {1 + n_{31}^{2}} \right)\sin^{2}\theta} - n_{31}^{2}} \right\rbrack}^{1/2}}}{and}} & (15) \\{E_{{0y},2}^{r} = \frac{2\cos \; \theta}{\left( {1 - n_{31}^{2}} \right)^{1/2}}} & (16)\end{matrix}$

In the equations immediately above, a thin film approximation is used,in order to greatly simplify the calculation of the field strength. Aspreviously mentioned, Harrick proposed this approximation. Therequirement to use this approximation is that the film must be very thinrelative to the depth of penetration if the sample were infinitelythick. The depth of penetration for a thick film at 6 μm measuringwavelength would be 2.32 μm. A monolayer of anthrax spores, for example,would have a thickness of approximately 0.4 μm, so the thin filmapproximation is valid for early detection and identification of anthraxspores deposited onto an ATR prism. The values used in the aboveequations are as follows: n₁=2.2, n₂=1.5, n₃=1.0, θ=45°, z_(i)=0. andz_(f)=0.4 μm. Calculated values for the field strength are as follows:E_(0x,2) ^(r)=1.37, E_(0z,2) ^(r)=0.79, and E_(0y,2) ^(r)=1.60.Calculated effective path for each vector are d_(ex) ^(iso)=0.45 μm,d_(ey) ^(iso)=0.62 μm, d_(ez) ^(iso)=0.15 μm, d_(e,∥) ^(iso)=0.60 μm,d_(e,⊥) ^(iso)=0.62 μm, and d _(e,random) ^(iso)=0.61 μm. The finalvalue for effective thickness is therefore 0.61 μm.

A single reflection through the ATR system modeled here would give riseto a signal (at 6 μm wavelength) that is comparable to a layer of sporesmeasured in transmission that is 0.6 μm thick, assuming a sporemonolayer with a thickness of 0.4 μm. So the ATR technique, even in asingle reflection, gives rise to a spectrum with 1.5× the strength of atransmission measurement. This figure can be increased dramatically byusing multiple reflections, making ATR infrared an excellent identifierof biological warfare agents such as anthrax.

Other concepts relating to ATR spectroscopy are disclosed in, forexample, U.S. Pat. No. 6,908,773 to Li et al. for ATR-FTIR Metal SurfaceCleanliness Monitoring; U.S. Pat. No. 7,218,270 to Tamburino for ATRTrajectory Tracking System (A-Track); U.S. Pat. No. 6,841,792 to Bynumet al. for ATR Crystal Device; U.S. Pat. No. 6,493,080 to Boese for ATRMeasuring Cell for FTIR Spectroscopy; U.S. Pat. No. 6,362,144 to Bermanet al. for Cleaning System for Infrared ATR Glucose Measurement System(II); U.S. Pat. No. 6,141,100 to Burka et al. for Imaging ATRSpectrometer; U.S. Pat. No. 6,430,424 to Berman et al. for Infrared ATRGlucose Measurement System Utilizing a Single Surface of Skin.

An often overlooked benefit of the ATR sampling mode for detecting andclassifying samples, however, is the immunity to the effects of scatter.Harrick notes that the ATR mode, unlike transmission or regularreflectance, removes the effect of light scatter. Even if a sample isgranular in nature, a situation that normally would give rise to lightscattering, the ATR spectrum will maintain a flat baseline. This meansthat different preparations of the same sample can be more similar toeach other, and therefore easier to classify in the same group. If thereexists real chemical differences between two samples, the differencesare more easily discerned because the sample morphology, preparation,and packing are removed as variables. An advantage of ATR, oftenoverlooked, is its immunity to the effects of scatter. A “perfect”infrared spectrum would contain only information related to themolecular structure of the sample. Sampling artifacts almost always aresuperimposed on this pure spectrum. However ATR can remove some of thedifferences due to sample scatter, improving the ability to identify andclassify a sample. This can be a huge advantage in the area of tissuespectroscopy.

An interesting recurring theme in the spectroscopy literature is theadmonition to stay away from the critical angle (Internal ReflectionSpectroscopy: Theory and Applications, Francis M. Mirabella, CRC Press,1993) because spectral distortions will result. This was noted early onin the seminal book by Harrick, and has been repeated many times since.The basis for this warning is seen in the depth of penetration equationslisted above. As the angle of incidence gets smaller and approaches thecritical angle, the depth of penetration of the evanescent wave into therarer medium gets larger and larger, up until the critical angle, atwhich point the total internal reflection condition no longer holds.Below the critical angle, internal reflectance turns into the much morecommon and much less useful external reflectance. External reflectanceis also governed by the laws of Fresnel reflection, but the resultingreflection is no longer total. In external reflection, it is notpossible to couple a large efficiency of energy back into the ATR prismand subsequently to the detector.

For many samples, it would be desirable to have a large depth ofpenetration into the sample. This could be achieved by introducingelectromagnetic energy very close to a critical angle for the sample. Inmost spectrometers, the light beam has a significant angular dispersion,in order to fill the detector and obtain high signal-to-noise ratio(SNR). However, because there is much angular dispersion, as thecritical angle is approached, a portion of the beam starts to exceed thecritical angle, while another portion of the beam is still at an anglethat is well away from the critical angle. In addition, in most samplesthere is dispersion in the refractive index across the spectral regionof interest, and so the critical angle is different for differentwavelengths. So these factors require the average angle to often beseveral degrees away from the critical angle.

It can be readily seen that the depth of penetration into the rarermedium can actually become quite large. There are many applications inwhich a larger depth of penetration would be desirable. The non-invasive measurement of body constituents is amongst these. Theteaching, repeated many times in the literature, is that ATR can nothave a large path length and can not have a large depth of penetration,because distortions of the spectrum occur near the critical angle. Thisproblem could be overcome by the use of a highly collimated beam oflight. Light sources are now available that can be highly collimated,yet still contain excellent amounts of energy. Many lasers such asquantum cascade lasers and light emitting diode (LED) sources are nowavailable that can be highly collimated and still contain large amountsof energy. But this is not a complete solution to the problem.

Another problem that needs to be overcome is the fact that most samplesthemselves exhibit wavelength dispersion in their refractive index. Ifuseful spectroscopic information about a sample is desired, whether byfluorescence, near infrared, terahertz, or some other spectroscopy, thesignal should be collected over some range of wavelengths. It willalmost certainly be true that over the wavelength range of interest, thecritical angle will vary with wavelength. The critical angle will evenchange within the same sample depending on various characteristics ofthe sample, such as the sample morphology or the physical state of thesample. Therefore it is very difficult, if not impossible to know, apriori, where the critical angle will lie, for a given sample at a givenwavelength. What is needed is an added dimension to the ATR measurement,namely that of a mapping of not only intensity versus wavelength, but ofintensity versus wavelength versus angle of incidence and/or reflection.

An ATR sampler can be designed that allows for multiple reflections.Multiple reflections thereby multiply the strength of the infraredspectrum. The number of reflections can be adjusted to arrive at anoptimum effective path length to give the highest possiblesignal-to-noise ratio. The apparatuses and methods described hereprovides for measurements that are at least one, and probably two,orders of magnitude more sensitive than making the measurement in atransmission mode or a traditional ATR mode. In order to successfullymap the angular space of interest, it would be desirable to cross overthe critical angle and also collect data below the critical angle. Thisdata could be useful in determining a true critical angle for eachwavelength.

SUMMARY OF THE INVENTION

An aspect of the invention is directed to an apparatus for detecting thespectral characteristics of a sample. The apparatus comprises anelectromagnetic radiation source adapted to excite a sample withelectromagnetic radiation; a crystal or prism in communication with theelectromagnetic radiation source and the sample, the crystal or prismhaving a high refractive index adapted to reflect the electromagneticradiation; a reflector adapted to introduce the electromagneticradiation to the sample at an angle of incidence at or near a criticalangle between the crystal or prism and the sample; and a detector fordetecting an electromagnetic radiation from the sample. Additionally thecomponents of the apparatus can be configured to be contained within ahousing. Suitable detectors for the apparatus include, but are notlimited to, a single element detector, such as a mercury telluridedetector, a linear array detector, and a 2-dimensional array detector.The electromagnetic radiation source can be adapted to deliver anelectromagnetic radiation to the sample at an angle of incidence whichis at or below the critical angle. In other configurations, theelectromagnetic radiation delivered to the sample can be delivered suchthat it approaches and passes the critical angle. In otherconfigurations, the radiation is delivered at an angle at or above thecritical angle. This radiation can also be adjusted to be delivered insuch a way that it approaches and passes the critical angle. Dataprocessors can also be provided that are in communication with thedetector. The data processors can be configured such that the dataprocessor receives information from any of the components of the systemand then generates a critical angle map of the sample from one or moreelectromagnetic radiation detections received by the detector from thesample. Suitable electromagnetic radiation sources include, for example,a quantum cascade laser. In some configurations, the apparatus isadapted to collimate the radiation. The apparatuses are configurable tobe housed in an area less than 1 cubic foot in volume, less than 125cubic inches in volume, and less than 8 cubic inches in volume. Suitableconfigurations are also adapted to be handheld. In other configurations,a display screen is provided. The display screen can be adapted andconfigured to display information useful to a user including, forexample, the critical angle map. The data processor can be adapted togenerate a full map of reflected light intensity versus wavelengthversus a mapping of the angle of incidence from the detectedelectromagnetic radiation. Moreover, in some aspects, a drive mechanismcan be provided. The drive mechanism can be adapted to pivot the crystalor prism about an axis. A cooler can also be provided. A cooler would beuseful for cooling the detector. Additionally one or more filters can beprovided and one or more lenses can be provided. Lenses can beconfigured to image the electromagnetic radiation onto a detector lessthat 1 mm squared.

Another aspect of the invention is directed to a method for detectingthe spectral characteristics of a sample. The method comprises, forexample, placing a sample in proximity to a crystal or prism; emittingan electromagnetic radiation from an electromagnetic radiation sourcethrough the crystal or prism; introducing the electromagnetic radiationto the sample through the crystal or prism at an angle of incidence ator near a critical angle of the sample; and detecting an electromagneticradiation from the sample. Additionally, the method can include thesteps of introducing the electromagnetic radiation at an angle ofincidence below the critical angle; and increasing the angle ofincidence of the electromagnetic radiation incrementally whereby theangle of incidence approaches and passes the critical angle. In someaspects of the method, the method can include the steps of introducingthe electromagnetic radiation at an angle of incidence above thecritical angle; and decreasing the angle of incidence of theelectromagnetic radiation incrementally whereby the angle of incidenceapproaches and passes the critical angle. Additionally, the method cancomprise or more steps of generating a full map of reflected lightintensity versus wavelength versus a mapping of the angle of incidence;displaying a generated map; comparing the detected electromagneticradiation to a database of critical angle measurements; displaying adetected electromagnetic radiation parameter and one or more criticalangle measurements from the database; filtering the electromagneticradiation; pivoting the crystal or prism about an axis; cooling thedetector; and imaging the electromagnetic radiation onto a detector arealess than 1 mm².

Still another aspect of the invention is directed to a system fordetecting the spectral characteristics of a sample. The systemcomprises, for example, an electromagnetic radiation source; a crystalor prism in communication with the electromagnetic radiation source andthe sample, the crystal or prism having a high refractive index adaptedto reflect the electromagnetic radiation internally; and a detector fordetecting an electromagnetic radiation from the sample. Additionally thecomponents of the system can be configured to be contained within ahousing. Suitable detectors for the system include, but are not limitedto, a single element detector, such as a mercury telluride detector, alinear array detector, and a 2-dimensional array detector. Theelectromagnetic radiation source can be adapted to deliver anelectromagnetic radiation to the sample at an angle of incidence whichis at or below the critical angle. In other configurations, theelectromagnetic radiation delivered to the sample can be delivered suchthat it approaches and passes the critical angle. In otherconfigurations, the radiation is delivered at an angle at or above thecritical angle. This radiation can also be adjusted to be delivered insuch a way that it approaches and passes the critical angle. Dataprocessors can also be provided that are in communication with thedetector. The data processors can be configured such that the dataprocessor receives information from any of the components of the systemand then generates a critical angle map of the sample from one or moreelectromagnetic radiation detections received by the detector from thesample. Suitable electromagnetic radiation sources include, for example,a quantum cascade laser. In some configurations, the system is adaptedto collimate the radiation. The systems are configurable to be housed inan area less than 1 cubic foot in volume, less than 125 cubic inches involume, and less than 8 cubic inches in volume. Suitable configurationsare also adapted to be handheld. In other configurations, a displayscreen is provided. The display screen can be adapted and configured todisplay information useful to a user including, for example, thecritical angle map. The data processor can be adapted to generate a fullmap of reflected light intensity versus wavelength versus a mapping ofthe angle of incidence from the detected electromagnetic radiation.Moreover, in some aspects, a drive mechanism can be provided. The drivemechanism can be adapted to pivot the crystal or prism about an axis. Acooler can also be provided. A cooler would be useful for cooling thedetector. Additionally one or more filters can be provided and one ormore lenses can be provided. Lenses can be configured to image theelectromagnetic radiation onto a detector less that 1 mm squared.

Kits are also contemplated as an aspect of the invention. Suitable kitsfor detecting the spectral characteristics of a sample, include, forexample, an electromagnetic radiation source; and a crystal or prism incommunication with the electromagnetic radiation source and the sample,the crystal or prism having a high refractive index adapted to reflectthe reflect the electromagnetic radiation. The kits can also includeother components, including, but not limited to one or more detectors,filters and/or lenses.

Incorporation by Reference

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a graph showing the correlation between incident angle and thedepth of penetration;

FIG. 2 is an illustration of a peri-critical reflectance spectroscopysystem;

FIG. 3 is an illustration of a peri-critical reflectance spectroscopysystem;

FIG. 4 is an illustration of a peri-critical reflectance spectroscopysystem showing imaging capability;

FIG. 5 is graph illustrating different effects achievable by changing anangle of incidence during spectroscopy;

FIG. 6 is an illustration of another peri-critical reflectancespectroscopy system wherein multiple reflections are achievable;

FIG. 7 illustrates a mechanism for changing an angle in a peri-criticalreflectance spectroscopy system;

FIG. 8 illustrates a 45 degree prism moving through various angles ofincidence; and

FIG. 9 illustrates an overview of a complete sampling system forspectroscopy.

DETAILED DESCRIPTION OF THE INVENTION

This invention therefore is directed toward the creation of devices andsystems that generate a critical angle map of a sample in addition to aspectral absorption map. The invention provides an added dimension tothe ATR measurement, by providing mapping of not only intensity versuswavelength, but of intensity versus wavelength versus angle of incidenceand/or intensity versus wavelength versus angle of incidence reflection.The devices and systems can be configured such that one or more elementsor components are formed integrally to achieve a desired physiological,operational or functional result such that the components complete thedevice. This can be achieved by one or more elements being integrallyformed as a single piece or being formed to act in a unified manner Theregion around the critical angle is a peri-critical region. Techniquesuseful to probe the peri-critical region include peri-criticalreflectance spectroscopy (PR).

Samples include, but are not limited to biological warfare agentdetection, non-invasive transcutaneous detection of glucose, ethanol,cancel cells, and other medically relevant constituents, biomarkers,drug components for new drug discovery, detection of explosives andother harmful chemical agents, early detection of infectious diseases,detection of trace chemical or biological contaminants in drinkingwater, illegal drug detection, determining the quality of industrialchemicals during production including biofuels such as biodiesel andbioethanol, determining the progress of reactions taking place inbioreactors, in vitro detecting and quantifying constituents of bloodsuch as glucose and creatinine The maps are generatable with highangular resolution near the critical angle for each wavelength. In mostinstances, the angular resolution is at least a millidegree or better.

I. DEVICES AND METHODS

A peri-critical reflectance spectroscopy apparatus or system, is adaptedto provide a source of electromagnetic radiation which can be introducedinto a sample, such as those described above. The electromagneticradiation can be modulated, for example, by an interferometer prior tocontacting the sample. The modulated radiation can also be focused by alens onto a reflective surface, such as a mirror, which then reflectsthe light into an ATR prism. Furthermore, in some instances, the mirrorcan be adjusted so that the electromagnetic radiation is introduced tothe sample through a range of angles which encompasses a target criticalangle. In other words, the electromagnetic radiation is introduced at anangle less than the critical angle and is swept in increments throughthe critical angle to an angle greater than the critical angle. Themirror can be adjusted to change the angle at which the electromagneticradiation enters the sample. Alternatively the electromagnetic radiationcan be introduced directly to the ATR prism. The electromagneticradiation, once inside the ATR prism then comes into contact with thesample. The electromagnetic radiation then exits the prism and isdetected by a detector and processed by a data processing system.

The critical angle information obtained using the systems and devicesdescribed herein is another dimension of information, which is not nowobtained with existing technology. A complete map of a sample wouldtherefore be a full map of reflected light intensity versus wavelengthversus a mapping of the angle of incidence, at angles that approach andthen in fact somewhat cross over, the critical angle. An angularresolution of a few millidegrees (a few microradians) is necessary,because, as illustrated in FIG. 1, the depth of penetration is verysensitive to the angle of incidence around the critical angle.Additionally, a processor can be used with the apparatus to analyze thecritical angle data. Once an angular map of the sample is generated by,for example, scanning the sample, the actual angle of the critical anglefor the each wavelength can be determined. A spectrum at each wavelengthat a constant effective depth can then be plotted.

FIG. 2 is an illustration of a peri-critical reflectance spectroscopysystem. A power source adapted and configured to provide power to asource 108 for electromagnetic radiation or light is adapted to delivera light beam to an interferometer 116, which separates the beam of lightinto two or more beams, such as by means of reflection, and thereafterbrings the rays together to produce interference. Suitable power sourcesinclude, but are not limited to, batteries. As will be appreciated bythose skilled in the art, the system can be contained within a suitablydesigned housing or the components can be configured such that thecomponents function as a housing. The resulting beam 110 then passesthrough a lens 150, after which it comes in contact with a mirror 130.The mirror reflects the resultant beam 112 through a prism 140 andtowards a sample 102. A reflected second beam 114 passes back throughthe prism 140 where it is received by a multi-element detector 160. Thedetector can be adapted and configured to resolve an angle of incidencefor the pixels to achieve a resolution of a millidegree or better. Theresolved pixels are then analyzed using a suitable data processingdevice or computer. The analysis can include, for example, comparing thedata against a library of data to determine a variance of the detectedsignal to a known sample. Additionally, the system can include adisplay, such as a liquid crystal display (LCD), adapted to provide adisplay to a user of the full map of reflected light intensity versuswavelength versus a mapping of the angle of incidence. As will beappreciated by those skilled in the art, connectivity can also beprovided which enables the system to sent the information to a printer,or a network. Connectivity can be, for example, wirelessly via theinternet as well as via suitable connection ports.

As will be appreciated by those skilled in the art, it is not alwaysnecessary to measure each angle discretely. The peri-criticalreflectance spectroscopy apparatus or system can be constructed as shownin FIG. 3. In FIG. 3, the spectroscopy apparatus 100 is set-up such thatthe electromagnetic radiation is introduced by a beam 110 to a sample102 using a mirror 130, such as a tilt/shift mirror having a 0.001degree resolution. The beam 110 can be delivered to the sample 102 afterbeing passed through a spatial filter 120. Passing the beam 110 throughthe filter 120 can result in a beam divergence, typically 0.001 degree.After passing through the filter 120, the divergence beam 122 comes incontact with a tilt shift mirror 130 which deflects the beam through aperi-critical reflectance (PR) crystal 140 into the sample 102. Suitablesamples can, for example, have a same area as low as 1-10 mm indiameter. After the beam comes in contact with the same, a resultingbeam 112 is reflected. The resultant beam 112 can then pass back throughthe PR crystal 140 to contact a second tilt/shift mirror 130′ whichtransmits the resultant beam 112 through a lens 150 and into a smallarea single element mercury cadmium telluride (MCT) detector 160.

A peri-critical reflectance (PR) spectroscopy instrument configured asshown in FIG. 3 can include a spatial filter 120 of variable size thatallows the infrared (IR) beam having one or more wavelengths from 750 nmto 1000 μm to be collimated to a desired angular resolution. Theresulting collimated beam has nearly parallel rays. As a result ofcollimating, a beam divergence of 1 millidegree is achievable. Launchmirrors 130, 130′ can then be configurable such that the mirrors cantilt and shift to vary the angle of incidence on the sample. Forexample, the angle can be varied in order to cross over a critical anglefor all wavelengths. A lens 150 can then be configured to image thespatial filter onto a very small detector area 162. Suitable areasinclude areas less than 1 mm², less than 0.01 mm², and more preferablyless than 0.001 mm² The small area detector enables sensitivityimprovement in systems that are limited by detector noise that usuallydominate during experiments in the mid-infrared spectroscopy range.

The system can also be adapted and configured such that the mirrorremains stationary relative to the sample. In such cases anelectromagnetic radiation source with a less collimated beam of energycan be used. Instead of sweeping the beam through a range of angles, theangular measurements can be made using a multiplicity of detectors(arrays) in such a manner such that each detector pixel element senses aprogressively smaller (or larger) angle, such angle to include thecritical angle at all wavelengths of interest. This detector array isdeployed after the sample and needs to be only a linear array ofdetector elements. Since it is often not possible to know beforehandwhat the critical angle will be at all of the wavelengths of interest, adetector containing a large number of pixels can be used. Otherwise, aspreviously described, the entire critical angle space could be mapped bysweeping the beam through different segments or portions of the totalcritical angle space in need of mapping.

In some instances the detector 160 can be cooled if desired for bettersensitivity. Cooling is achievable using a suitable cooling apparatus,means for cooling, or material. For example, cooling with liquidnitrogen may, in some instances, improve sensitivity of the detector.Cooling typically involves decreasing the temperature of the detectorsemiconductor material to the temperature of liquid nitrogen and mostpreferably to the temperature of liquid helium.

The beam generated by the system may be an output beam of a FourierTransform Infrared (FTIR) spectrometer. However, as will be appreciatedby those skilled in the art, a beam from a single or series of quantumcascade (QC) lasers may also be used. In some instances, selection of abeam type or source can improve the portability of the devices orsystems. Thus, for example, a system less than 1 cubic foot in volumecan be transported easily, and a system less than 125 cubic inches involume can be handheld, and a system less than 8 cubic inches in volumemay be concealed and hidden from view. This scalability of size providessignificant advantages. Moreover, QC lasers can be highly collimated.

Alternative to using a multi-element detector, the angle of incidence ofthe beam may be changed manually and successive scans made. The inputand the output angle may be changed together in order to obtain acomplete map of the spectral data at the entire range of angle ofincidences.

The angles interrogated should extend both above and below the expectedcritical angle. This is because the critical angle varies as a functionof wavelength. The goal is to re-create a spectrum as a constant andknown degree of closeness to the critical angle, or constant effectivedepth. In this manner, spectral distortions normally associated withworking too close to the critical angle are completely obviated. It isnow possible to collect undistorted spectra, while working very close tothe critical angle. This allows the ATR method to have longer pathlength and deeper penetration into the rarer medium (the sample undertest) than is possible using conventional methods. This will beparticularly important in non-invasive biological measurements and manyother measurements such as: detection of low levels of biologicalwarfare agents.

Turning to FIG. 4, an illustration of a peri-critical reflectancespectroscopy system is provided showing imaging capability. In thisembodiment, the crystal 140/sample 102 surface is imaged onto an ArrayMCT detector 160 instead of the spatial filter, as illustrated above.The previous single element detector is replaced with a one- ortwo-dimensional detector array, as desired. A two-dimensional detectorarray can be adapted and configured to collect hyperspectral data withone dimension of wavelength, 2 dimensions of image and the further onedimension of angle of incidence. Each of these dimensions can, as willbe appreciated by those skilled in the art, have thousands of datapoints. The depth of profiling capability of this system and techniqueallows for the creation of a three-dimensional spatial profile of asample volume with spectral information at each spatial position. Themultiple detectors have the effect of reducing the time needed tocollect a data set, directly in proportion to the number of detectorelements. Additionally a sample 102 area of 1-10 mm in diameter can beused.

FIG. 5 is graph illustrating different effects achievable by changing anangle of incidence during spectroscopy. Light rays are launched with ahigh index, or dense medium. Well below the critical angle(sub-critical), light refracts at the crystal/sample interface and thenmostly transmits into the sample itself as a propagating wave. If thesample is scattering, then diffuse reflectance (DR) is the result. Wellabove the critical angle (super critical) light reflects totally and aweak standing or evanescent wave is set up in the rare medium (sample).As a result, no light waves propagate in the sample. The characteristicsof the resulting sample spectrum is consistent with attenuated totalreflectance (ATR) Immediately in the vicinity of the critical angle(peri-critical), light becomes very sensitive to angle. At thecrystal/sample interface, three things happen: light reflects at thenegative critical angle, a strong evanescent wave is set-up in thesample, and a traveling wave propagates in a direction parallel to thecrystal sample interface plane. This effect benefits peri-criticalreflectance (PR) spectroscopy. By resolving angles accurately, to amillidegree, it is possible to map the peri-critical region for allwavelengths and refractive indices present in the given sample andcrystal. The reflected PR beam contains strong information about thesample and from deeper depths into the sample than is possible by ATR.

Turning now to FIG. 6, an illustration of another peri-criticalreflectance spectroscopy system wherein multiple reflections areachievable is provided. A beam 110 from an electromagnetic radiationsource passes through a negative lens 152 and hits a first mirror 132.The beam 110 is deflected from the first mirror 132, forming a resultantbeam 112. The resultant beam 112 then hits a second mirror 134 and formsa second resultant beam 114, which comes in contact with a peri-criticalreflectance crystal or prism 140. The second resultant beam passesthrough the PR crystal from which it is then passes through a negativelens 154. Multiple reflections are achieved which are all at or near thecritical angle. A precision drive (not shown), or any suitable means tomove or rotate the platform, causes a platform to rotate or move. Theplatform carries first mirror 132, second mirror 134, and the PR crystal140. The drive enables, for example, the platform to pivot around apivot point 144 situated at or near an exit face 146 of the crystal 142.The negative lenses 152, 154 allow the instrument to be used in thesample compartment of many FTIR spectrometers that have a focusing beamnear the center of the sample compartment. An example of a suitable FTIRdevice would be any Thermo Nicolet FTIR (Thermo Fisher Scientific,Waltham Mass.). The negative lenses collimate the beam, allowing angularresolution of the resulting collimated beam. Beam divergence, canfurther be limited by the J-stop (Jacquinot stop or field stop) insidethe spectrometer, usually near the source. The beam divergence of theelectromagnetic or IR beam is determined by an angular measurement of anincrease in beam diameter over a distance from the source, or opticalaperture.

As illustrated in FIG. 7, a mechanism for changing an angle in aperi-critical reflectance spectroscopy system can be achieved. A 45degree prism 142 made of a high index crystal, such as zinc selenide(ZnSe), can be used. The beam is launched in and out of the bottom face142′ of the prism 142 such that a first beam 110 enters the bottom face142′ of the crystal and a second beam 110′ departs the bottom face 142′of the crystal parallel or substantially parallel to the first beam 110.The internal reflections of the beam occur at two facets of the prismfollowing the path illustrated by the dashed line. Thus the incomingfirst beam 110 perpendicularly enters the bottom face 142′ of thecrystal, hits a facet of the prism where it is deflected at an angle of90 degrees. The deflected beam then hits a second facet within the prismwhere it is deflected a second time at an angle of 90 degrees. Thesecond deflected beam 110′ then exits perpendicularly through the bottomface 142′ of the crystal such that the incoming first beam 110 anddeparting second beam 110′ are substantially parallel. The prism can betilted about a pivot point 144. As a result of tilting the prism 142^(T) around the pivot point, the angle of incidence on one facetincreases while the angle of incidence on the other facet decreases. Thesample under test can be adjacent to, or adhered to, one facet or theother of the prism. The input and output beams remain parallel to eachother as the prism tilts. The ability to tilt the crystal whileretaining parallel beams minimizes a need for realignment where there isan angle change.

Turning now to FIG. 8, a 45 degree prism shown on a top line movingthrough five separate sample angles of incidence (A-E). The prismretains the parallel beams as shown in the bottom line and as describedabove. Thus, an angular range of up to 10 degrees or more can be usefulin PR spectroscopy.

FIG. 9 illustrates an overview of a complete sampling system forspectroscopy. The sampling system employs the previously described 45degree prism. In this configuration, a pivot point is found that makesthe input and output beams remain stationary during crystal tilting. Aswill be appreciated by those skilled in the art, the crystal is depictedwith long facet and a short facet.

II. KITS

Kits are also contemplated as an aspect of the invention. Suitable kitsfor detecting the spectral characteristics of a sample, include, forexample, an electromagnetic radiation source; and a crystal incommunication with the electromagnetic radiation source and the sample,the crystal having a high refractive index adapted to reflect thereflect the electromagnetic radiation. The kits can also include othercomponents, including, but not limited to one or more detectors, filtersand/or lenses.

III. EXAMPLES Example 1 Determining Blood Glucose Levels

The devices and methods described above can be uses to detect levels ofglucose. The skin surface of a patient can be placed in proximity to thesystem. Thereafter, the skin is radiated with an electromagneticradiation beam through the transmitting crystal. A beam is reflectedback out and through the crystal. The return beam carries with itinformation indicating the blood glucose level in the user. The returnbeam can be analyzed using a suitable word processor to provide, forexample, a full map of reflected light intensity versus wavelengthversus a mapping of the angle of incidence. This information can becorrelated with other biological parameter information. Additional themap can be displayed on and LCD and/or communicated to a network.

Example 2 Non-Contact Inspection of Materials

Another application is in the area of non-contact inspection. Normallywith ATR, it is essential to create a very intimate optical contactbetween the ATR crystal and the specimen under test. Without thisintimate contact, an intermediate layer, usually air, must be consideredin the refractive index and depth calculations. With powders and otherirregular samples, it is often impossible to remove all of the airspace. As a result, the measurement is often unstable from onemeasurement to the next. The other reason for intimate optical contactis that since the depth of penetration is so small in ATR, the goal isto get the specimen as close to the crystal as possible, where theevanescent field is the strongest. With the present invention, it ispossible to make the depth of penetration much larger. Therefore we canget very good spectra even when the specimen is not in physical contactwith the ATR crystal. The problem of instability in the region of theevanescent field is thereby avoided. An excellent application of this isin the area of non-contact inspection of materials, especially when thematerial is moving, for instance on a production line. A particularapplication in the non-contact inspection field would be the examinationof pharmaceutical tablets on a production line.

REFERENCES

J. Fahrenfort, Spectrochim. Acta 17, 698 (1961).

Harrick, N. J., Internal Reflection Spectroscopy, New York: WileyInterscience, 1967.

Fringeli U P, Goette J, Reiter G, Siam M, and Baurecht D (1998)Structural Investigations of Oriented Membrane Assemblies by FTIR-ATRSpectroscopy. In Proceedings of the 11^(th) International Conference onFourier Transform Spectroscopy.

Messerschmidt R G, Multiple Internal Reflectance Spectroscopy System,U.S. Pat. No. 4,730,882 (1988).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-20. (canceled)
 21. A method for detecting the spectral characteristicsof a sample, comprising: placing a sample in proximity to a crystal;serially introducing electromagnetic radiation from an electromagneticradiation source to the crystal at a plurality of angles such that theelectromagnetic radiation intersects a measurement site of the samplethrough a crystal at a plurality of angles of incidence, wherein theangles of incidence are incrementally changed between each introductionof the electromagnetic radiation; detecting a reflected electromagneticradiation from the sample at the plurality of angles of incidence with adetector; and generating an angular map of the sample, wherein theangular map comprises a mapping of reflected electromagnetic radiationintensity relative to the plurality of angles of incidence.
 22. Themethod of claim 21, wherein the step of introducing the electromagneticradiation comprises introducing the electromagnetic radiation at a firstangle of incidence; and incrementally increasing the angles of incidenceof electromagnetic radiation delivered to the sample to approach asecond angle of incidence.
 23. The method of claim 21, wherein the stepof introducing the electromagnetic radiation comprises introducing theelectromagnetic radiation at a first angle of incidence; andincrementally decreasing the angles of incidence of electromagneticradiation delivered to the sample to approach a second angle ofincidence.
 24. The method of claim 21, further comprising the step ofdetermining a critical angle between the crystal and the sample.
 25. Themethod of claim 21, further comprising the step of determining a depthof penetration of an evanescent wave into the sample at the plurality ofangles.
 26. The method of claim 25, further comprising the step ofgenerating a depth spectrum of evanescent wave absorption.
 27. Themethod of claim 21, wherein the step of generating the angular mapfurther comprises plotting reflected electromagnetic radiation versuswavelength relative to a mapping of the angles of incidence.
 28. Themethod of claim 21, further comprising the step of comparing thereflected electromagnetic radiation to a database of critical anglemeasurements.
 29. The method of claim 26, further comprising the step ofdisplaying a detected electromagnetic radiation parameter and one ormore critical angle measurements from the database.
 30. The method ofclaim 21, wherein the step of emitting electromagnetic radiation from anelectromagnetic radiation source further comprises collimating theelectromagnetic radiation to have a beam divergence of one millidegreeor less.
 31. The method of claim 21, wherein the step of introducing thecollimated beam further comprises introducing the collimated beam to thesample through the crystal with an angular resolution of one millidegreeor better.
 32. The method of claim 21, further comprising the step ofimaging the reflected electromagnetic radiation onto a detector arealess than 1 mm².
 33. The method of claim 21, further comprising the stepof modulating the electromagnetic radiation prior to introducing theelectromagnetic radiation to the sample.
 34. The method of claim 31,further comprising the step of focusing the modulated electromagneticradiation onto a reflector, wherein the reflector is adapted tointroduce the electromagnetic radiation to the sample.
 35. A systemcomprising: at least one processor; and a computer-readable mediumstoring one or more sequences of instructions which, when executed bythe at least one processor, causes: serially introducing electromagneticradiation from an electromagnetic radiation source to a crystal at aplurality of angles such that the electromagnetic radiation intersects ameasurement site of the sample through a crystal at a plurality ofangles of incidence, wherein the angles of incidence are incrementallychanged between each introduction of the electromagnetic radiation;detecting a reflected electromagnetic radiation from the sample at theplurality of angles of incidence with a detector; and generating anangular map of the sample, wherein the angular map comprises a mappingof reflected electromagnetic radiation intensity relative to theplurality of angles of incidence.
 36. The system of claim 35, whereinthe instructions that cause introducing the electromagnetic radiationcomprises instructions that cause introducing the electromagneticradiation at a first angle of incidence; and incrementally increasingthe angles of incidence of electromagnetic radiation delivered to thesample to approach a second angle of incidence.
 37. The system of claim35, wherein the instructions that cause introducing the electromagneticradiation comprises instructions that cause introducing theelectromagnetic radiation at a first angle of incidence; andincrementally decreasing the angles of incidence of electromagneticradiation delivered to the sample to approach a second angle ofincidence.
 38. The system of claim 35, wherein the instructions thatcause generating the angular map further comprises instructions thatcause plotting reflected electromagnetic radiation versus wavelengthrelative to a mapping of the angles of incidence.
 39. The system ofclaim 35, further comprising instructions which, when executed by the atleast one processor, causes determining a depth of penetration of anevanescent wave into the sample at the plurality of angles.
 40. Thesystem of claim 35, further comprising instructions which, when executedby the at least one processor, causes comparing the reflectedelectromagnetic radiation to a database of critical angle measurements.